This invention relates to a method of detecting hematopoietic progenitor cells (HPCs). The method is capable of simple counting of peripheral blood HPCs for use as transplants in PBSCT (peripheral blood stem cell or PBSC transplantation) and it provides a useful index for determining the right time to collect PBSCs. PBSCs are the most primitive fraction of HPCs and are considered the “origin” or stem cells of all hematopoiesis. As will be explained specifically below, the term “hematopoietic progenitor cells” as used herein is synonymous with but broader in sense than the term “peripheral blood stem cells”.
Bone marrow transplantation (BMT) has seen a substantial development over the past 30 years as the last resort in the treatment of malignant diseases such as leukemia, lymphoma, myeloma and certain solid tumors. In spite of the many advantages it has, BMT also involves various difficulties such as the need to perform multiple bone marrow apheresis while patient or donors are under on general anesthesia. Hence, the feasibility of this technique is limited to either university hospitals or other major medical institutions of a comparable class and there exist many hospitals that do not practice BMT.
On the other hand, the recent advances in the studies of hematopoietic stem cells and the introduction of pharmaceutical preparations containing hematopoietic factors as an active ingredient have made it possible to mobilize HPCs within the peripheral blood. This, combined with the remarkable improvements in the technology of blood component separators, has contributed to the rapidly increasing use of PBSCT.
BMT and PBSCT share the common feature of transplanting stem cells but PBSCT has the following advantages over BMT:                (1) the donor need not be placed under general anesthesia when extracting stem cells but simple apheresis will suffice, which contributes to safety;        (2) more rapid hematopoietic recovery when compared with BMT is possible after the transplantation and, hence, the risk of infection and the need for blood transfusions is reduced;        (3) the period of hospitalization is shortened;        (4) a large number of stem cells are included in the transplant;        (5) contamination by tumor cells is limited; and        (6) the incidence of death associated with the transplatation is low.        
The general procedure of PBSCT is as follows. First, the patient is administered chemotherapy, hematopoietic growth hormones (interleukins e.g. granulocyte colony stimulating factor (G-CSF)) and any other necessary reagents, whereupon the leukocyte count in the peripheral blood initially decreases while on the subsequent day 5-7 or later, the leukocyte count starts to increase. Similarly, but not necessarily concurrently with this event, the number of hematopoietic stem cells also increases.
When a sufficiently large number of hematopoietic stem cells have been mobilized in the peripheral blood (usually in about 5-20 days), the stem cells are collected by means of a blood component separator and stored frozen. In this instance, it is necessary to know exactly when the number of stem cells in the peripheral blood is increased. The timing of mobilization and collecting of the hematopoietic stem cells should be neither too early nor too late in order to secure an adequate number of stem cells.
In the next step, the cancer cells are killed by applying very high levels of chemotherapy and/or radiotherapy that are sufficient to destroy the patient's bone marrow. Thereafter, the previously collected stem cells are transplanted so as to achieve rapid recovery of the hematopoietic capability of the patient.
As will be understood from the foregoing, successful PBSCT requires efficient collection of hematopoietic cells and, to this end, the mobilization of peripheral stem cells has to be monitored correctly. Two common methods currently used to count stem cells are a colony assay method and a CD34 positive (a marker which is present on the cell surface of HPC) cell count method using flow cytometry (FCM). In the colony assay method, the number of stem cells to be collected is adjusted appropriately in a suitable culture medium such as IMDM and cultivation is performed in a blood stem cell assay medium for 14 days in a CO2 incubator while a colony count is obtained with a phase-contrast microscope. In the CD34 positive cell count method using FCM, particularly in single-color analysis, a whole blood sample is reacted with a fluorescence-labelled anti-CD34 monoclonal antibody and, after hemolysis flow cytometry is performed and a CD34 positive cell count is obtained from a scattergram of lateral scattered light and flourescence or a histogram of fluorescence. In a two-color analysis, flow cytometry is performed using an anti-CD34 monoclonal antibody and an anti-CD45 monoclonal antibody and the CD45 positive cells alone are first incorporated as data, which are then analyzed using lateral scattered light and CD34 fluorescence as parameters such that cells are counted which produce low levels of lateral scattered light and which express CD34.
The colony assay method is the currently the most accurate way to obtain the correct PBSC count and this is sometimes used to perform the stem cell count on samples that have been subjected to apheresis. A problem with this method is the long time (2 weeks) that is required to complete the measurement and, therefore, it is not suitable to determine the right timing for HPC/PBSCs collection. In addition, while requiring a high level of skill on the part of the operating personnel, the colony assay method is not a technique that assures good reproduction of the results and which can be adopted in routine work. High operating cost is another problem with this method.
Compared to the colony assay method, the CD34 positive cell count method can be completed in a short time but it still takes at least 1-2 hours in the counting procedure. For monitoring the mobilization of stem cells within the peripheral blood, the measurement is ideally continued for 3-5 days or more. However, using a flow cytometer in order to count CD34 positive cells, the method under consideration is difficult to adopt as a routine technique on account of the high cost of equipment and reagents, as well as the scarcity of skilled engineers. In addition, gating is necessary to determine specific cell zones on a scattergram or histogram and this produces considerable differences from one facility to another.
Given these circumstances, a blood cell count is often adopted in clinical practice and the degree of mobilization of HPC is monitored indirectly by looking at the pattern of increase in the number of leukocytes and platelets and the right time to collect stem cells is determined on the basis of these results, assuming a state of mobilization of HPC. Some people argue that this method suffcies for clinical purposes but it is obvious from many observation that the method lacks consistency. Stated more specifically, if the leukocyte count increases in synchronism with the platelet count, sometimes this helps to determine the right time to collect HPC; however, this is often not the case, especially if there is a mismatch between the rates of increase of the two counts.
Recently, an automatic blood cell analyzer equipped with channels capable of detecting immature leukocytes (IMI, or immature leukocyte information, channel) has been introduced into the market as Model SE-9000 from Toa Medical Electronics Co., Ltd. and many reports were thereafter published on the results of PBSC counting using this model; examples are as follows: Lebeck, L. K. et al., International Society for Laboratory Hematology, Vol.1, No.1, p.62, 1995; Takekawa, K. et al., The Japanese Journal of Clinical Hematology, Vol.36, No.9, 1995; Yamada, H. et al., Japanese Journal of Medical Technology, Vol.145, No.3, p.501, 1996; Mougi, H. et al., Med. J. Kagoshima Univ., Vol.48, No.2, p.139-146, 1996; Houwen, B. et al., International Society for Laboratory Hematology, Vol.2, No.2, p.51, 1996; and Takekawa, K. et al., Journal, of the American Society of Hematology, Vol.88, No.10, Supplement 1, 250b, 1996. According to these reports, a statistically significant correlationship has been found to exist between the total count of immature leukocytes (IMI total) and the number of CD34 positive cells as determined with a flow cytometer.
In fact, however, the IMI total includes the cells that appear in the zones for blasts, immature granulocytes and left shifts and the PBSC count is not the only parameter that is reflected by the IMI total. In other words, the IMI total which includes the above-mentioned rather mature juvenile leukocytes involves such substantial errors that it is not suitable for use in monitoring the appearance of PBSCs that can be adopted in PBSCT.